The present invention relates to a process for determining the total protein content or individual amino acids in flour, cereal flour and/or leguminous products by means of fluorescence analysis, the proteins and/or the individual amino acids being treated to attain a fluorescent effect or are themselves fluorescent, as well as to an apparatus for practicing the method.
In the breeding of plants with the aim of increasing the quantity and quality of proteins, it is necessary to develop fast and accurate analysis methods to determine the total protein content or individual amino acids therein. This necessity is a result of the poor food situation in the world. The previously employed methods, which are substantially of the chemical type or are activation analytic, have been found to be too slow, inaccurate and are connected with substantial amounts of work. In the prior methods, a total protein determination is generally effected by initially making a chemical or activation analytic nitrogen determination and then calculating the total protein content via numerical factors. This calculation, however, also includes nitrogen which is not bound to proteins.
In the majority of determination methods employed in plant breeding, e.g. for determining lysine, the proteins are hydrolized. An exception is the dye binding capacity method (DBC). In the methods where the proteins are hydrolized, the flour samples are generally decomposed in constantly boiling 6N hydrochloric acid. Alkali and enzymatic hydrolysis can also be used. In addition to the great expenditures of time and money, the hydrolysis step also produces errors from incomplete separation of the amino acids, and a racemization and destruction of part of the amino acids. The hydrolyzate can be examined as to its lysine content by a series of different tests.
Chromatographic and electrophoretic determination methods have been used but are expensive and require practical experience. Since they furnish the total composition of the proteins, however, they are of advantage for the breeder who also wants to be informed about other essential amino acids. The separation of the hydrolyzate can be effected by paper chromatography, column chromatography or electrophoresis. The quantitative determination of the individual amino acids is usually effected colorimetrically after dying with ninhydrine. See, for example, S. Blackburn, "Amino Acid Determination" Marcel Dekker, Inc. New York (1968).
Another prior art method which involves hydrolysis is the FDNB method which is based on the reaction of fluoro-2, 4-dinitrobenzol (FDNB) with free amino groups of proteins and peptides in alkali solution. After the hydrolysis, dinitrophenyl amino acid is obtained which includes .epsilon.-DNP-lysine. The quantitative determination of the latter is effected colorimetrically. See, for example, K. J. Carpenter, Biochemical Journal, 77 (1960) 604.
In still another prior art determination method, bacteria have been grown for enzymatic determination which produce specific decarboxylases of lysine and other amino acids (E. F. Gale, Advances in Enzymology 6 (1946) 1). L-lysine decarboxylases are obtained from bacterium cadaveris. Manometric determination of the CO.sub.2 provides the lysine content of the hydrolyzate. This method also is expensive and time consuming.
In a further prior art determination method based on biologic determination, an essential amino acid is withheld from the nutrient medium of a bacteria culture so that growth will stop. After addition of an amino acid, hydrolyzate growth is proportional to the proportion of the amino acid lacking in the nutrient medium. This method is simple and very specific but does not attain high reproduceability (A. E. Bolinder, Acta Pharm. Suec. 7, (1970) 125).
For the routine lysine determination in cereal flours, the already-mentioned dye binding capacity method (D. C. Udy, Cereal Chemistry 33 (1956) 191) has found widest acceptance. In the dye binding capacity method, the sample to be examined is ground and mixed with a dye solution of orange G (7-hydroxy-8-pheylazonaphthaline disulfonic acid-(1,3)-disodium salt) of known concentration. THe dye reacts with the basic amino acids, lysine, histidine and arginine. After draining of the dye, the concentration of the unbound dyestuff in the filtrate is determined. The difference in concentration leads to a conclusion regarding the quantity of basic amino acids. It is clear that the values obtained in this way have a high correlation to the total protein content of the flour samples. The total protein content might be determined by a nitrogen determination according to Kjeldahl where 16 g nitrogen correspond to approximately 100 g protein. The advantage of the DBC method is in the avoidance of hydrolysis. It is therefore simple, quick and furnishes easily reproduceable results. Its drawback, however, is that it is not specific, e.g. for lysine. High DBC values may also be caused by high proportions of arginine and histidine and a low lysine content.
A fluorometric determination of basic and total proteins by means of sulfoflavine is also known (U. Leemann and F. Ruch, Journal of Histochemistry, Cytochemistry, 20, 659-671, 1972). This fluorometric analysis method could be transferred, with some modifications, from histochemistry and cytology to application in connection with plant breeding, yet the known fluorometers are too complicated technically for such purposes.
For example, measurements are made with a spectral fluorometer "Fluorispec" made by the firm Baird-Atomic, Model SF 100. The initial or excitation radiation is the light from a high pressure xenon lamp. In order to spectrally divide the excitation and fluorescence radiation, two double monochromators of the Czerny-Turner type with dispersion ranges of 220 nm to 700 nm are used. Each one contains two planar reflection grids. The blaze angles are designed for 300 nm on the excitation side and for 500 nm on the emission side. The fluorescence radiation is measured at a right angle to the direction of impingement of the excitation radiation.
The curves recorded by this fluorometer, however, do not represent the true fluorescence spectra of the substance to be examined. Rather, they are falsified by the wavelength dependent sensitivity of the photomultiplier employed and by the selective effect of the emission monochromator (blaze angle). Analogously, the excitation spectra are distorted by the nonconstant spectral intensity distribution of the high pressure xenon lamp and by the selectivity of the excitation monochromator. For evaluation, the measured spectra generally must be corrected. A prerequisite for breeding success is therefore, for example, that the lysine content or the tryptophan content of the seed material be determined with sufficient speed and precision during the various selection states. The above-mentioned known fluorometer is unnecessarily complicated and expensive for these protein and/or amino acid determinations in cereal flours. Moreover, sedimentation of the flour would begin and would lead to considerable measuring errors as a result of the horizontal path of the beams in the instrument.